1. Technical Field
A transcription regulatory region, comprising a nucleotide sequence, which promotes early seed-specific transcription of contiguous nucleotide sequences is provided.
2. Background
A large number of genes are known which are expressed only in developing seeds, or are expressed in developing seeds at much higher levels than in any other organ or tissue type. For the purposes herein, xe2x80x9cgene expressionxe2x80x9d refers to synthesis of mRNA corresponding to a given gene. Thus, the amount of gene expression generally refers to the rate of transcription, or the rate of synthesis of the mRNA. For convenience, in the context of this invention, we have generally assumed that differences in the steady-state level of mRNA accumulation reflects differences in the rate of synthesis of the mRNA. It is understood that in some cases changes in the steady-state level of mRNA could be caused by changes in the rate of mRNA degradation. However, it is considered unlikely that manipulation of promoter sequences, as taught herein, will generally affect the rate of mRNA degradation.
Much of the information about seed-specific gene expression has been derived from studies of genes encoding storage proteins (reviewed by Bevan et al., 1993). For instance, DNA sequences that confer embryo-specific expression by the soybean conglycinin promoter in transgenic plants have been identified (Chen et al., 1988). Similarly, the storage protein napin is one of the major protein components of Brassica napus L. (oilseed rape) seeds. A 152 bp fragment from the napin promoter directed strong expression of the xcex2-glucuronidase reporter gene in mature tobacco seeds (Stalberg et al., 1996). Thus, the sequences that direct strong seed-specific expression of storage proteins are conserved between distantly related plant species. The napin promoter has been used to control expression of genes in transgenic plants designed to produce novel fatty acids (e.g., Voelker et al., 1996). However, because storage lipid accumulation begins substantially before the maximal level of expression of the napin or other storage protein genes is reached (Post-Beittenmillar et al., 1992), the promoters of storage protein genes may not be preferred for controlling expression of genes related to storage lipid accumulation.
In the present invention, a preferred regulatory region (e.g., promoter, enhancer, silencer) for expression of genes directed toward modification of seed lipid composition, or other applications, would be derived from a gene that has a similar, or identical, temporal and tissue-specific pattern of expression to the genes that encode enzymes involved in seed storage lipid synthesis and accumulation. However, until recently, relatively few genes were known which are involved in lipid metabolism and are expressed in seed-specific manner. The kappa hydroxylase from the Crucifer Lesquerella fendleri is one of the first examples of this class of genes. A promoter of the present invention normally controls the expression of the kappa hydroxylase from L. fendleri. Kappa hydroxylase is thought to be located in the endoplasmic reticulum where it catalyzes the introduction of a hydroxyl group into fatty acids attached to the sn-2 position of phospholipids. Since hydroxylated fatty acids are abundant in the seed storage lipids of L. fendleri but are not found to any appreciable extent in other organs or tissues, it seems likely that the gene is only expressed appreciably in seeds. The isolation of the kappa hydroxylase gene from L. fendleri was described in U.S. patent application Ser. Nos. 08/530,862 and 08/597,313, and PCT/US97/02187. Evidence was presented showing that the mRNA for the kappa hydroxylase was abundant in seeds but was not detectable in vegetative tissues. Here, we demonstrate that a regulatory region in the 5xe2x80x2 direction from the coding sequence of the L. fendleri kappa hydroxylase is useful as a seed-specific promoter in plant species other than L. fendleri. We also show that the regulatory region can be used to cause early seed-specific expression of a gene other than the kappa hydroxylase (i.e., heterologous gene expression) Regulatory regions having the desired properties described herein should also be found upstream of other plant fatty acyl hydroxylase genes isolated and identified as disclosed in the parent applications.
It is an object of the present invention to provide a transcription regulatory region for use in transgenic plants that exhibits seed-specific expression of a gene product.
It is a further object of the present invention to provide a transcription regulatory region for use in transgenic plants that exhibits an early level of expression of a gene product in developing seeds.
It is yet another object of the present invention to provide a transcription regulatory region for use in transgenic plants that exhibits high-level expression of a gene product.
Isolated nucleic acids (e.g., DNA, RNA, cDNA, cRNA) are provided that comprise a transcription regulatory region (e.g., promoter, enhancer, silencer) from a plant fatty acyl hydroxylase gene. Preferably, the plant gene is a kappa hydroxylase gene and, more preferably, the kappa hydroxylase gene is from Lesquerella. The regulatory region may comprise a nucleotide sequence from SEQ ID NO:1. The regulatory region may comprise between about 500 nucleotides to about 2000 nucleotides, and may be capable of directing expression at a high level, at an early stage of development, in a seed-specific manner, or a combination thereof. The isolated nucleic acid may further comprise a sequence encoding for the native fatty acyl hydroxylase.
Recombinant nucleic acids (e.g., DNA, RNA, cDNA, cRNA) are provided that are comprised of the isolated nucleic acid described above and an operably linked non-native sequence to be transcribed. The sequence may be from the same plant species from which the regulatory region is derived or from a different species or genera; the sequence may even be from a bacterial, fungal, or mammalian gene. Preferably, the sequence is derived from a plant gene, especially one that is involved in seed lipid metabolism or seed development. The sequence may be in the sense or antisense orientation relative to transcription.
Expression constructs (e.g., DNA, RNA, cDNA, CRNA) are provided which are employed in manipulating plant cells to provide for early and/or seed-specific transcription. In particular, transcription regulatory regions from a gene encoding a fatty acyl hydroxylase are operably linked to other than the native or homologous gene, and introduced into a plant cell host for integration into the genome to provide for early and/or seed-specific transcription. The constructs provide for modulation of expression of endogenous products as well as production of exogenous products in the seed.
Transformed host cells, transgenic plants, and transgenic seeds are provided that contain an integrated or non-integrated nucleic acid, recombinant nucleic acid, or expression construct as described above. The host cell may be of bacterial, fungal, plant, animal, or similar origin. The transgenic plant may be Arabidopsis, Brassica, cotton, soybean, safflower, sunflower, tobacco, flax, peanut, or any other dicot species in which early seed-specific gene expression is desired. The regulatory region of the invention may also be useful in controlling seed-specific expression of genes in monocotyledonous species such as wheat, maize, rice, or the like. Transgenic seeds may be derived from similar plant species. Oil may be pressed, or otherwise extracted, or other materials such as proteins, carbohydrates, polyalkanoates, or secondary metabolites may be extracted from the transgenic seed.
Kits are also provided containing a nucleic acid, recombinant nucleic acid, expression construct, host cell, or a combination thereof with directions for the use of the aforementioned to produce a transgenic plant or seed.